1. Field of the Invention
The present invention relates to variant DNA ligases. In particular, the invention relates to modified thermostable DNA ligases having higher DNA binding activity and enhanced reaction activity compared to the wild type, which can be obtained by substitution of a negatively charged amino acid present at the N-terminal side of the C-terminal helix moiety of the DNA ligase with a non-negatively charged amid acid, the DNA encoding the modified thermostable DNA ligases, and the use of such modified thermostable DNA ligases, and the like.
2. Background Art
DNA ligase is an enzyme having activity that links a 3′-hydroxyl group of a DNA chain to a 5′-phosphate group of a DNA chain via a phosphodiester bond, and plays a role in replication and repair of DNA in vivo. In recent years, the ligase chain reaction (LCR) method has been developed as a new gene amplification technique, and used. The LCR method is a method of amplifying or detecting a target gene through a thermal cycling reaction using a thermostable DNA ligase. Ligases with improved thermostability have been sought for improving the efficiency of LCR method, and some are commercially available. Very recently, a DNA ligase from a hyperthermophilic archaeon has been found (as described in Non-Patent Document 1). However, these thermostable DNA ligases have a very low DNA binding activity. DNA ligases from phages are known as enzymes with high DNA binding activity. But they are not suited for use in the LCR method because of their poor thermostability. Thus, a thermostable DNA ligase with good DNA binding activity and reactivity, with which the LCR method can be carried out efficiently at a satisfactory reaction rate, has not yet been found.    [Non-Patent Document 1]: “Successful development of a genetic diagnostic enzyme (DNA ligase) having the highest known thermal stability”, Press release on the website of the National Institute of Advanced Industrial Science and Technology (2003)